WO1996002636A1

WO1996002636A1 - Genes encoding and method of expressing a novel enzyme: phthalyl amidase

Info

Publication number: WO1996002636A1
Application number: PCT/US1995/008919
Authority: WO
Inventors: Stephen Wyatt Queener; Joseph Martin Zock
Original assignee: Eli Lilly And Company
Priority date: 1994-07-15
Filing date: 1995-07-14
Publication date: 1996-02-01
Also published as: IL114593A0; ZA955899B; CA2194783A1; US5451522A; EP0769051A1; JPH10502818A; YU47895A; EP0769051A4; US5543497A; CO4480058A1; PE32696A1; AU3099895A

Abstract

Phthalyl amidase is an enzyme previously unknown in the art that catalyzes removal of the phthalyl moiety from phthalyl-containing amides. The current invention provides DNA compounds encoding the phthalyl amidase enzyme and methods for expressing such compounds. The present invention also provides recombinant DNA vectors encoding phthalyl amidase and host cells transformed with these DNA vectors.

Description

Title

GENES ENCODING AND METHOD OF EXPRESSING A NOVEL ENZYME:

PHTHALYL AMIDASE Background of the Invention

The present invention relates to the discovery of a specific enzyme that has not been previously

described, a phthalyl amidase, which readily removes the phthalyl moiety from phthalyl-containing amides. The present invention also relates to an organism isolated from natural sources that produces the enzyme, DNA compounds -that encode the enzyme, and methods for producing and using the enzyme.

The phthalimido functional group is an important tool in organic synthesis because of the protection it provides against unwanted reactions. However,

dephthalylation reactions generally require harsh

conditions and often have low yields thereby limiting the situations in which phthalimido protection can be employed.

Removal of a phthalyl protecting group from a phthalyl amide can be accomplished chemically, Kukolja et al., Croatica Chemica Acta 49:779, 1977, but yields are variable especially with substrates that are unstable to harsh reaction conditions.

Certain enzymes have previously been found that could be used to remove benzoyl groups from benzoylated amino acids. Toyoura et al., Chem. Pharm. Bull. 7:789, 1959. These enzymes were specific for benzoyl groups and for the amino acid to which they were attached. Others have also reported enzymes that will hydrolyze phthalate esters. Kurane et al., Agric. Biol. Chem. 44:529, 1980. However, none of these enzymes have been shown to operate on phthalyl amides.

In contrast, the phthalyl amidase enzyme of this invention catalyzes removal of the phthalyl group from a wide variety of phthalyl-containing compounds with improved yields over processes known in the art, exhibits

stereochemical selectivity, and eliminates the need for harsh conditions to remove the protecting group.

Summary of the Invention

The present invention provides a recombinant phthalyl amidase enzyme, which catalyzes the following type of reaction:

The present invention also provides DNA compounds that comprise isolated nucleotide sequences encoding the phthalyl amidase enzyme and methods for expressing such compounds. The present invention also provides recombinant DNA vectors encoding phthalyl amidase and host cells transformed with these DNA vectors. Preferred DNA compounds comprise an isolated nucleotide sequence encoding SEQ ID NO:2, especially SEQ ID NO: 1 isolated from Xanthobacter agilis . Other preferred

compounds of the present invention include DNA compounds that comprise isolated DNA sequences encoding the proenzyme form of phthalyl amidase enzyme (SEQ ID NO:4), including SEQ ID NO: 3, SEQ ID NO: 5, and the phthalyl amidase gene of Xanthobacter agilis (SEQ ID NO:6). DNA compounds of the current invention include recombinant DNA vectors,

including expression vectors, which may be used to

transform host cells.

The present invention also provides for DNA sequences of the naturally-occurring phthalyl amidase gene that control transcription, translation, and extra-cellular secretion of proteins. Thus, the present invention includes SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO: 10.

Definitions:

Coding sequence - the sequence of DNA in the open reading frame of a gene that encodes the amino acid residue sequence of the protein expressed from the gene.

Gene - a segment of DNA that comprises a

promoter, translational activating sequence, coding sequence, and 3' regulatory sequences, positioned to drive expression of the gene product.

Promoter - a DNA sequence that directs or initiates the transcription of DNA. Recombinant DNA vector - any autonomously replicating or integrating DNA agent, including but not limited to plasmids, comprising a promoter and other regulatory sequences positioned to drive expression of a DNA sequence that encodes a polypeptide or RNA.

Recombinant DNA sequence - any DNA sequence, excluding the host chromosome from which the DNA is

derived, which comprises a DNA sequence that has been isolated, synthesized, or partially synthesized.

Restriction fragment - any linear DNA molecule generated by the action of one or more restriction enzymes.

Translation activating sequence - a regulatory DNA sequence that, when transcribed into mRNA, promotes translation of mRNA into protein.

All nucleotide and amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R.

S1.822(b)(1993). Brief Description of the Figures

The restriction enzyme and function maps

presented in the drawings are approximate representations of the recombinant DNA vectors discussed herein. The restriction site information is not exhaustive. There may be more restriction enzymes sites of a given type than are actually shown on the map.

Figure 1 is a restriction enzyme site and function map of plasmid pZPA600. Abbreviations: PAorf = phthalyl amidase open reading frame, tsr = gene enabling resistance to thiostrepton.

Figure 2 is a restriction enzyme site and function map of plasmid pZPA400. Abbreviations: PA-orf = phthalyl amidase open reading frame. P197-pro = modified promoter from phage lamda. tet = gene enabling resistance to thiostrepton. C1857 = gene encoding temperature

sensitive lamda repressor. Detailed Description of the Invention

During the course of developing a chiral, shorter, and more efficient synthetic route to loracarbef ([6R-(6A,7B(R))]-7-[(aminophenylacetyl)amino]-3-chloro-8- oxo-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid), the Mitsunobu reaction (see e.g. Hughes, D.L. Organic reactions 42:336, 1992; Bose, A.K. et al., Can. J. Chem. 62:2498, 1984) was selected for forming the beta-lactam ring from a chiral linear amino acid ester intermediate. Several reactants with one N-valence protected and a few reactants with both N-valences protected were examined in Mitsunobu reactions. They were either not cyclized or were cyclized in poor yield.

It was discovered that problems in forming the beta-lactam ring via Mitsunobu reactions could be overcome if both valences of the α-nitrogen of the chiral linear amino acid ester intermediate were protected with a phthalimido group. However, no known chemical reaction was available to remove the phthalimido moiety and regenerate free amine in high yield.

Thus, soil samples were examined for microorganisms that could catalyze removal of the

phthalamido group from a test substrate (II) that was formed by base cleavage of the phthalimido ring of a bivalently N-protected compound. A culture was identified that demonstrated phthalyl amidase activity that liberated the free amine derivative of the test substrate. Native enzyme was purified and shown to catalyze the following desired reaction:

Phthalyl amidase also has significant value in peptide synthesis applications. Phthalimido amino acid derivatives are very effective reactants for enzymatic coupling of amino acids to form peptides. However, heretofore, methods for removing the phthalimido blocking group from the protected peptide were lacking. The phthalyl amidase of the current invention displays reactivity toward a wide range of substrates and can be used for deblocking phthalimido-protected peptide

intermediates.

The isolated phthalyl amidase of this invention demonstrates high specific activity toward phthalylated amides or esters (i.e., having a 1,2 dicarboxylate

configuration). Such compounds may have other functional groups on the phthalyl aromatic ring and still serve as substrates for the enzyme. For example, acceptable

functional groups include 6-F, 6-NH₂, and 3-OH. Moreover, substrates may include a nitrogen in the aromatic ring ortho to the carboxyl group attached to the amine.

Compounds lacking a 2-carboxylate, such as benzoyl, phenylacetate, phenoxyacetate, or their derivatives, are not suitable substrates for this enzyme.

The enzyme also exhibits a broad substrate specificity in regard to the amine group attached to the phthalate side chain. For example, phthalylated amino acids and peptides, mono- and bicyclic beta-lactams, aromatic and non-aromatic amines, as well as phthalylated amines attached to heterocycles, are dephthalylated by this enzyme at acceptable catalytic rates. The enzyme also removes the methyl group from mono-methyl phthalate.

The enzyme is stable in the broad range of pH from 6-9, having an optimum stability pH of 8.0 ± 0.4. The enzyme also demonstrates a marked stability dependence on ionic strength. Ionic strength above 20 mM enhances pH and temperature stability of the enzyme. Optimum ionic

strength occurs at 200 mM and above.

The enzyme retains good activity in low salt (50 mM) up to 30° C and in high salt (200 mM) up to 40° C. In 200 mM salt, at least 80% of the enzyme activity is retained in temperatures up to 35°C for 48 hours.

Iodoacetic acid (10 mM), p-HMB (1 mM), and Cu⁺⁺ (1 mM) significantly inhibited the enzyme. No organic co- factors, such as ATP, NADPH, or others, stimulated enzyme activity. EDTA, phenanthroline, and metal ions besides Cu⁺⁺ had little or no effect on enzyme activity.

The molecular weight of the enzyme is approximately 49,900, as determined by electrospray mass spectrometry, and the molecule consists of one subunit.

The K_m, with phthalamido carbacephem (7- phthalamido-3-chloro-4-carboxy-1-carba-dethioceph-3-em) (III) as substrate, is 0.9 mM in 50 mM potassium phosphate buffer, pH 8.0, and 30° C. The V_max for this substrate and under these conditions is 7.6 μmol/min/mg.

Phthalyl amidase activity was recovered from a microorganism isolated from soil samples. The organism was characterized by comparison of its fatty acid methyl ester profile with that of known standards, and has been

identified as a strain of Xanthobacter agilis .

The organism can be preserved as lyophilized culture and has been deposited with the National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois 61604-39999, under accession number NRRL B-21115 (date of deposit: 7/16/93). Working cultures are maintained as liquid cultures stored in liquid nitrogen or at temperatures below -78°C.

In order to recover the phthalyl amidase of this invention, Xanthobacter agilis can be cultivated in an aqueous nutrient medium consisting of a source of carbon and nitrogen and mineral salts at an initial pH between 6 and 8 and at 25° to 37° C. A number of agents can be included in the culture medium as inducers of enzyme production, including, for example, phthalate (PAA), phthalyl glycine (PAG), and phthalyl monocyclic beta-lactam (PMBL). The enzyme can be recovered in larger amounts by cultivating Xanthobacter agilis in a known manner in a bioreactor of desired size, for example, with a working volume of 100 liters. Good aerating conditions, and the presence of nutrients in complex form, and a pH between 6 and 8 are important for a successful culture. The cell mass can be separated from the medium and the enzyme purified as shown in Example 4.

It will be recognized by those skilled in the art that phthalyl amidase-producing mutants of the isolated Xanthobacter agilis organism can readily be made by methods known in the art. These mutants are considered within the scope of this invention.

As described, phthalyl amidase, catalyzes the removal of the phthalyl moiety from a wide range of phthalimido-containing compounds. The enzyme actually cleaves the amide bond of a phthalamido substrate, which is formed by the action of mild base on the corresponding phthalimido compound. This conversion proceeds readily under conditions that are suitable for enzyme activity. Thus, the phthalimido-containing compound and the enzyme being concurrently present under conditions that promote enzyme activity result in in situ removal of the phthalyl group.

In some chemical reactions involving an amine reactant, the corresponding phthalimido compound is particularly suited to high reaction yields whereas the conversion proceeds poorly with the unprotected amine or with a monovalently protected amine or even when the amine is bivalently protected by an alternative means. Thus, the current invention, which provides an economic source of phthalyl amidase, allows practical synthesis of a variety of amine products via phthalimido-protected amine

intermediates.

It will be recognized that the enzyme can also be used in immobilized form to catalyze desired reactions according to procedures known in the art.

A specific application of the current invention occurs in a new chiral synthesis of the antibiotic

loracarbef. The phthalyl amidase-catalyzed reaction shown above is one step of that synthesis.

Another application occurs in the synthesis of aspartame (N-L-α-aspartyl-L-phenylalanine, 1-methyl ester) as described in Example 16 below. In both cases phthalic anhydride (or other suitable activated forms of phthalic acid) is used to react with an intermediate containing a key amino group so that a phthalimido moiety is formed for bivalent protection of the amino group. The bivalently protected amine can then be converted efficiently to a desired intermediate. For example, cyclization of a α-phthalimido-β-hydroxy-acid to a beta-lactam, or for example, condensation of an α- phthalimido carboxy-activated amino acid with a carboxy- protected amino acid to form a dipeptide. The phthalimido moiety is hydrolyzed with mild base and the resulting phthalamido moiety is then exposed to phthalyl amidase to catalyze the removal of the phthalyl moiety and release free amine plus phthalic acid.

In addition to identification and isolation of a naturally-occurring phthalyl amidase, the current invention provides DNA compounds that comprise an isolated nucleotide sequence encoding phthalyl amidase, recombinant DNA vectors encoding phthalyl amidase, host cells transformed with these DNA vectors, and a method for producing recombinant phthalyl amidase. These elements of the current invention provide the opportunity to use phthalyl amidase as a biocatalyst in industrial scale chemical processes.

Phthalyl amidase may be produced by cloning DNA encoding phthalyl amidase into a variety of vectors by means that are well known in the art. A number of suitable vectors may be used, including cosmids, plasmids,

bacteriophage, and viruses. One of the principle requirements for such a vector is that it be capable of reproducing itself and transforming a host cell.

Preferably, the vector will be a recombinant DNA vector that is capable of driving expression of phthalyl amidase encoded by the DNA compounds of this invention. Typical expression vectors comprise a promoter region, a 5'- untranslated region, a coding sequence, a 3'-untranslated region, an origin of replication, a selective marker, and a transcription termination site.

After the DNA compound encoding phthalyl amidase has been inserted into the vector, the vector may be used to transform a host cell. In general, the host cell may comprise any cellular organism, including a prokaryotic cell or eukaryotic cell, that is capable of being

transformed with a vector comprising the DNA of this invention. The techniques of transforming and transfecting cells are well known in the art and may be found in such general references as Maniatis, et al . (1989) or Current Protocols in Molecular Biology (1989).

A particularly preferred method of the current invention generates soluble, extra-cellular enzyme. The method makes use of a DNA compound that comprises SEQ ID NO: 6, which enables, when transformed into Streptomyces lividans as part of a self-replicating vector, the host to produce and secrete soluble mature phthalyl amidase in an amount in excess of the amount of a cell-bound form of the enzyme produced by Xanthobacter agilis , the bacterium from which the DNA compound was cloned. SEQ ID NO: 6 comprises four functional components: SEQ ID NO:7; SEQ ID NO: 8; SEQ ID NO: 1; and SEQ ID NO: 10. SEQ ID NO: 7, which includes the promoter-bearing nucleotides 1-135 of SEQ ID NO: 6, promotes transcription of the coding sequences. SEQ ID NO: 8 (nucleotides 136-261 of SEQ ID NO: 6) encodes the signal peptide portion of a proenzyme form of phthalyl amidase (pro-phthalyl amidase (SEQ ID NO:4)). The signal peptide (SEQ ID NO: 9), which provides for transport of the proenzyme across the

microbial cell wall of Streptomyces lividans, is cleaved from the proenzyme by the cell, thereby enabling extracellular production of the mature enzyme. SEQ ID NO: 1 (nucleotides 262-1620 of SEQ ID NO:6) encodes mature phthalyl amidase (SEQ ID NO:2). SEQ ID NO:10 (nucleotides 1621-3029 of SEQ ID NO:6) is a 3'-untranslated region which assists proper and efficient translation termination of the mRNA that encodes pro-phthalyl amidase.

Moreover, in a more general application of the expression method of the current invention, a wide variety of soluble, extra-cellular, properly-folded, functional proteins may be produced in Streptomyces . The current method comprises propagating Streptomyces lividans that has been transformed with a DNA compound, which encodes the desired enzyme, protein, or peptide, and which includes the transcriptional and translational regulatory elements of the phthalyl amidase gene isolated from the bacterium Xanthobacter agilis . These regulatory elements enable synthesis and secretion of the soluble, properly-folded, functional enzyme, protein, or peptide.

To accomplish the general method, the DNA sequence encoding mature phthalyl amidase (SEQ ID NO: 1) may be replaced in SEQ ID NO: 6 by a heterologous open reading frame from a wide variety of organisms wherein the

heterologous open reading frame encodes a mature protein or hormone and introns are absent from those open reading frames, either by nature or by virtue of precise removal from genomic DNA to form cDNA open reading frames. In this arrangement, the regulatory elements of the phthalyl amidase gene continue to function such that proteins and oligopeptides other than phthalyl amidase are produced and secreted from Streptomyces transformed with the modified DNA sequence. Thus, substitution of a desired protein- encoding sequence for the coding sequence of mature phthalyl amidase enables economic extra-cellular production of numerous enzymes, peptides, and peptide hormones.

Synthesis of the phthalyl amidase gene and its various elements can be accomplished by recombinant DNA technology. Synthetic genes, the in vitro or in vivo transcription and translation of which will result in the production of the phthalyl amidase enzyme, may be

constructed by techniques well known in the art. Owing to the degeneracy of the genetic code, the skilled artisan will recognize that a sizable, yet definite, number of DNA sequences may be constructed, which encode the phthalyl amidase enzyme. All such sequences are provided by the present invention.

A preferred sequence encoding phthalyl amidase is the naturally-occurring phthalyl amidase gene of

Xanthobacter agilis, which is SEQ ID NO: 6. This preferred gene is available on an 3.2 kb SacI-BamHI restriction fragment of plasmid pZPA600, which can be isolated from Streptomyces lividans TK23/pZPA600 by techniques well known in the art. Streptomyces lividans TK23/pZPA600 designates Streptomyces lividans strain TK23 which has been

transformed with vector pZPA600.

Plasmid pZPA600 was derived by ligating SEQ ID NO: 6 into Streptomyces vector, pIJ702 (Hopwood, D.A., et al., Genetic Manipulations of Streptomyces : A Laboratory Manual , The John Innes Foundation, Norwich, England, 1985). The pIJ702 vector contains a pIJ101 Streptomyces replicon and a thiostrepton resistance gene for selection. The ligated material was transformed into Streptomyces lividans TK23 by a standard protoplast fusion technique. After selection on thiostrepton (45 μg/ml), the plasmid

designated pZPA600, was isolated and confirmed by

restriction analysis. A restriction site and function map of plasmid pZPA600 is found in Figure 1.

Streptomyces lividans TK23/pZPA600 is publicly available and on deposit at the National Center for

Agricultural utilization Research, 1815 North University Street, Peoria, Illinois 61604-39999, under accession number NRRL 21290 (date of deposit: 6/23/94). The Streptomyces lividans TK23 strain has been previously described in Plasmid 12:1936 (1984).

Plasmid pZPA600 allows high level expression of the pro-phthalyl amidase open reading frame and results in secretion of soluble mature phthalyl amidase, which process is especially preferred. Thus, the invention comprises a process in which Streptomyces lividans TK23/pZPA600 is grown and then separated from its extra-cellular broth so that high concentrations of phthalyl amidase are obtained in that cell-free broth.

Other preferred sequences include, for example, SEQ ID NO: 1, which encodes mature phthalyl amidase enzyme (SEQ ID NO:2), and SEQ ID NO: 3, which encodes the proenzyme form of phthalyl amidase (SEQ ID NO:4). Thus, the present invention also comprises plasmid pZPA400 as a preferred embodiment.

In plasmid pZPA400, the 5'- regulatory elements of the native gene were removed and an ATG codon for a methionyl residue was attached to the 5'-terminal

nucleotide of the mature phthalyl amidase coding sequence to generate an open reading frame (SEQ ID NO: 11) encoding met-phthalyl amidase (SEQ ID NO: 12). SEQ ID NO: 11 was positioned, via a two-cistron configuration, such that transcription was driven by a temperature inducible lambda pL promoter. Plasmid pZPA400 also contains the temperature sensitive cI857 repressor gene, a tetracycline resistance gene, and the pBR322-based origin of replication minus the rop region, which controls copy number (Cesareni et al., Proc. Natl. Acad. Sci. 79:6313, 1982). E. coli cells harboring this plasmid ( E. coli DH5α/pZPA400) are induced to produce met-phthalyl amidase (without signal peptide) when the culture temperature is raised from 30° C to 42° C. A restriction site and function map of plasmid pZPA400, which can be isolated from E. coli DH5α/pZPA400 cells by techniques well known in the art, is found in Figure 2.

E. coli DH5α/pZPA400 designates the commercially available E. coli DH5α strain that has been transformed with plasmid pZPA400. E. coli DH5α/pZPA400 cells are publicly available and on deposit at the National Center for Agricultural Utilization Research, 1815 North

University Street, Peoria, Illinois 61604-39999, under accession number NRRL B21289 (date of deposit: 6/23/94).

The phthalyl amidase gene may also be created by synthetic methodology. Such methodology of synthetic gene construction is well known in the art. See Brown et al. (1979) Methods in Enzymology, Academic Press, N.Y., 68:109. The phthalyl amidase DNA sequence may be generated using a conventional DNA synthesizing apparatus, such as the

Applied Biosystems Model 380A of 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404.

Synthesis of the phthalyl amidase protein of the present invention may also proceed by solid phase

synthesis. The principles of solid phase chemical

synthesis of polypeptides are well known in the art and may be found in general texts, such as, Dugas, H. and Penny, C., Bioorganic Chemistry (1981), Springer-Verlag, New York, pp. 54-92. However, recombinant methods are preferred if a high yield is desired.

A skilled artisan will recognize that the nucleotide sequences described in the present disclosure may be altered by methods known in the art to produce additional sequences that substantially correspond to the described sequences without changing their functional aspects. These altered sequences are considered to be included in the current invention.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that the examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

EXAMPLE 1

Search for phthalyl amidase producing organisms

240 soil samples (8 to 15 mg of damp dry soil) were individually suspended in 10 ml sterile BL medium (hereinafter defined) containing 100 mg phthalyl monocyclic beta-lactam (PMBL)

BL medium had the following composition:

The cultures were incubated aerobically at 30° C in a rotary shaker at 250 rpm for as long as 2 weeks.

Cultures were examined by thin layer chromatography at 7 day intervals for the disappearance of the starting substrate and appearance of the beta-lactam nucleus product.

A culture showing the desired catalytic activity was transferred at least two more times under similar conditions of medium and growth. The final culture was diluted with sterile water and plated out on agar plates containing either Trypticase Soy Broth (Difco) or Bac MI medium. Bac MI medium had the following composition:

(Agar plates were prepared by adding 20 g agar per L of medium).

Individual colonies were picked from the agar and grown in Bac MI medium containing 10 mg/ml of PMBL for 12 days at 30°C with aeration. Broths were examined for appearance of beta-lactam nucleus and phthalic acid using TLC.

A pure isolated organism that demonstrated rapid hydrolysis of the substrate was then grown in Bac MI medium containing 1 mg/ml phthalate for 48 hours at 30° C with aeration. Cells were centrifuged and then suspended in 50 mM Tris-HCl buffer, pH 8.0, at a ratio of 1 g wet weight cells to 8 ml of buffer. A solution of lysozyme, 2 mg in 1.0 ml 50 mM EDTA, pH 8.2, was added at the ratio of 1 ml lysozyme solution to 8 ml cell suspension. After mixing well and holding at room temperature for 1 hour, the suspension was cooled to 4° C and held overnight. The resultant viscous solution was sonicated only long enough to liquefy the solution. This solution was centrifuged at 10,000 rpm for 15 minutes. The pellet was discarded and the supernatant tested for phthalyl amidase activity.

The cell-free extract was chromatographed on a size exclusion column (1.5 x 100 cm; Sephacryl S-300;

Pharmacia, Piscataway, NJ) at 4° C with an elution buffer consisting of 50 mM potassium phosphate and 150 mM KCl at a flow rate of 0.5 ml/min. The eluant was monitored at a wavelength of 280 run. UV-absorbing fractions were tested for hydrolysis of PMBL by HPLC.

Reference proteins for molecular weight (daltons) determination were chymotrypsinogen (25,000), ovalbumin (43,000), albumin (67,000), aldolase (158,000), catalase (232,000), ferritin (440,000), and thyroglobulin (669,000).

Cell-free extract of the organism subsequently identified as Xanthobacter agilis was determined to contain an enzyme that catalyzed the hydrolysis of PMBL, and which had an approximate molecular weight of 54,000 daltons and a specific activity of 39.7 nmol/min/mg.

EXAMPLE 2

Production of phthalyl amidase from Xanthobacter agilis

Fermentation of Xanthobacter agilis on a 100 L scale was conducted in 100 L working volume bioreactors, with automatic control for pH (7.9-8.1), temperature (30° C), air flow (1 scfm), agitation (300 rpm), and back pressure (5 lb). Dissolved oxygen levels (>50%) were kept constant by small increases in agitation speed. The medium consisted of 1.25% Bacto peptone, 0.3% yeast extract, 0.5% beef extract, 0.5% phthalic acid, 0.5% NaCl, and 0.05% anti-foam. After sterilization, the medium was brought to pH 8.0 with 30% sulfuric acid. The fermenter was

inoculated with 1 L of pre-culture which had been incubated at 30° C for 24 hours m the same medium with shaking at 300 rpm. After 48 hours of growth, the fermentation broth was cooled and centrifuged at 17,000 rpm with a flow rate of 1 to 2 L/min to remove the biomass. The cell paste was harvested and stored at -20° C yielding 6.0 g wet cell weight/L.

EXAMPLE 3

Induction of phthalyl amidase

Three compounds at different concentrations were added to aerated cultures of the organism growing at 30° C in Bac MI medium. The compounds tested were phthalate (PAA), phthalyl glycme (PAG), and PMBL Ceils of

Xanthohacter agilis were grown with aeration for 24 hours. This vegetative culture was used to inoculate Bac MI medium (50 ml) containing different concentrations of the

compounds to be tested. After 48 hours growth under standard conditions, cells were harvested by centrifugation and wet weight of the cells was determined. Crude cell extracts were prepared by lysozyme treatment of the cells as in Example 1. Suspensions were briefly sonicated to break up the viscous suspension. A cell-free supernatant was obtained by centrifugation of the suspension at 14,000 rpm for 15 minutes.

Enzyme activity in cell-free lysates was determined by monitoring conversion of the chromogenic substrate 4-(2'-carboxy-N-benzoyl) amino-2-carboxy- nitrobenzene (II) to 2-nitro-5-amino benzoic acid and phthalic acid, a reaction catalyzed by phthalyl amidase as shown below:

The assay reaction mixture (1 ml) consisted of

0.3 μmol of the chromogenic substrate (II) and 0.001-0.5 μg of enzyme preparation in 50 mM potassium phosphate buffer, pH 8.0 (buffer A). The enzymatic reaction was conducted at 30° C for 10 minutes and the appearance of product was monitored at 380 nm (or 430 run). The amount of substrate hydrolyzed was calculated from a standard curve of the amine product.

As can be seen in Table 1, PAG and PAA increased the wet weight cell mass while PMBL had no effect.

However, all three substrates produced a dramatic

concentration-dependent increase in the total number of enzyme units recovered. The units of enzyme per gram of wet weight cells also increased with all additions but the increase was most pronounced at high PAA concentrations.

EXAMPLE 4

Purification of phthalyl amidase

A. Analytical scale purification of phthalyl amidase

Cells of Xanthobacter agilis (200 grams, wet weight), which contained significant amounts of phthalyl amidase activity, were resuspended to 1800 ml in 50 mM Tris-HCl, pH 8.0, plus 5 mM EDTA. The cells were broken by sonication for 22 minutes at a maximal power below 8° C. DNase (1 μg/ml) and magnesium sulfate (10 mM) were added during the sonication to minimize viscosity and improve cell breakage. After a high-speed centrifugation, the resulting crude extract supernatant served as the source for further enzyme purification. All subsequent

purification steps were conducted at 4° C.

The crude extract was loaded onto a Q-Sepharose column (4.4 x 23 cm; Pharmacia), previously equilibrated with buffer A. After washing with buffer A, a linear gradient of 0-1.5 M KCl in buffer A was applied and the phthalyl amidase eluted as a single activity peak between 1 and 1.1 M KCl. Selected fractions containing most of the enzyme activity were pooled as Q-Sepharose eluate.

The Q-Sepharose eluate was subjected to ammonium sulfate fractionation. The majority of the enzyme activity was recovered from 67-77, 77-87 and 87-97% ammonium sulfate pellets. Those pellets were solubilized in buffer A with 0.2 M ammonium sulfate. Ammonium sulfate was added to the 67-97%

ammonium sulfate enzyme pool to a final concentration of approximately 2 M. The enzyme pool was loaded onto a Phenyl-Sepharose column (2.6 x 16 cm; Pharmacia), which was previously equilibrated with buffer A plus 2.6 M ammonium sulfate. The phthalyl amidase eluted with a linear gradient decreasing from 2.6 M to 0 M ammonium sulfate in buffer A as a single activity peak between 0 M and 0.5 M ammonium sulfate. Selected fractions containing the majority of the enzyme activity were pooled as Phenyl- Sepharose eluate.

The Phenyl-Sepharose eluate was dialyzed against buffer A and then loaded onto a hydroxylapatite column (1.5 x 90 cm; Clarkson Chemical Company, Williamsport, PA), which was previously equilibrated with buffer A. After washing the column with buffer A, the enzyme eluted with a linear gradient of 50-500 mM potassium phosphate, pH 8.0, as a single activity peak between 150 and 190 mM potassium phosphate. Selected fractions containing most of the enzyme activity were pooled as hydroxylapatite eluate.

After a dilution of the buffer strength from 120 to 50 mM potassium phosphate, the hydroxylapatite eluate was loaded onto a Mono P column (0.5 x 20 cm; Pharmacia), which was previously equilibrated with buffer A. After washing with 3 column volumes of buffer A, a linear gradient of 0-1.5 M KCl in buffer A was applied and the enzyme eluted as a single activity peak between 0.72 and 0.8 M KCl. Those fractions containing the majority of the enzyme activity were pooled as Mono P eluate. The most active enzyme preparation was derived from Mono P FPLC (Fast Protein Liquid Chromatography).

Table 2 surnmarizes the results of the purification. Based on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Laser Densitometric Scanning, the phthalyl amidase was greater than 95% pure.

The phthalyl amidase activity reported in Table 2 was determined using the chromogenic substrate as in

Example 3. A typical reaction mixture in a total volume of 1 ml contained 0.2 mg of the chromogenic substrate and an aliquot of phthalyl amidase in buffer A. The enzymatic reaction was conducted at 30° C for 10-15 min. Formation of the reaction product was monitored with a

spectrophotometer at 430 nm (or 380 nm) and quantitated from a standard curve of the product.

B. Preparative scale purification of phthalyl amidase

Crude extract of Xanthobacter agilis was

prepared by adding 1 g of cells (wet weight) and 2 mg lysozyme per 9 ml of 50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA (600 g cells total). After 30 minutes at room temperature, DNase (100 U/g of cells) in 10 mM magnesium sulfate was added. The cells were homogenized using a cell homogenizer for 30 minutes at room temperature. After 17 hours of incubation at 8° C, the lysate was centrifuged at 10,000 rpm for 30 minutes. The crude extract supernatant (4.5 L) was applied to a Super-Q column (7 x 40 cm; TosoHaas,

Montgomeryville, PA) equilibrated in buffer A. After loading crude extract, the column was washed with 2 column volumes of 50 mM phosphate buffer containing 3.5 M urea, pH 8.0. A second wash (5 L) was used to re-equilibrate the column in buffer A. Phthalyl amidase eluted from the column using a 10 column-volume linear gradient of 0-1.5 M KCl in buffer A. Fractions were collected and assayed for enzyme activity. The active fractions were pooled (1.5 L), concentrated (250 ml), and diafiltered with buffer A at 7- 10° C.

The concentrated and diafiltered Super-Q mainstream was applied to a hydroxylapatite column (3.2 x 40 cm) equilibrated in buffer A. After washing the column with this buffer, phthalyl amidase was eluted using a linear gradient of 0-500 mM phosphate buffer, pH 8.0.

Fractions were assayed according to the chromogenic

substrate method (see Example 3) and the active fractions were pooled (I D and concentrated (400 ml).

Table 3 shows the results of this purification.

EXAMPLE 5

Effect of pH on phthalyl amidase activity

The effect of pH on the reaction rate of the analytical scale purified enzyme was determined using phthalamido carbacephem (III) as substrate.

A typical reaction mixture consisted of 1 ml total volume and contained 0.1 mM III, 0.1 μM phthalyl amidase in 50 mM potassium phosphate buffer (pH 5.5-9.0) at 32° C for 20 minutes. The reactions were stopped by the addition of 1 ml methanol. After removal of precipitate by centrifugation, an aliquot of the supernatant fraction (typically 30 μl) was monitored for the beta-lactam nucleus and phthalic acid by HPLC using a Zorbax C8 column (0.46 x 15 cm; MacMod Analytical Inc., Chadds Ford, PA). The two reaction products were eluted by a mobile phase constructed as continuous mixed gradients from (a) 1% ACN

(acetonitrile) /0.2% TFA (trifluoroacetic acid) and (b) 80% ACN/0.2% TFA as follows: 1) 0% (b), 3 min; 2) 0-50% (b), 0.5 min; 3) 50-100% (b), 3 min; 4) 100% (b), 2.5 min; 5) 100-0 % (b), 0.1 min; and 6) 0% (b), 5 min. At a flow rate of 1.5 ml/min, retention times of the beta-lactam nucleus and phthalic acid, as measured at 254 nm, were 2.3 and 7.2 min, respectively.

The results are shown in Table 4. Optimal range for enzyme activity occurred between pH 7.8 and 8.4.

EXAMPLE 6

Optimum reaction temperature

Test reactions were carried out similar to

Example 5 except that all incubations were performed m 50 mM potassium phosphate buffer at pH 8.2. Solutions of the substrate were pre-incubated for 5 minutes at temperatures between 2 and 60° C. The enzymatic reaction was initiated by the addition of phthalyl amidase and stopped by the addition of 1 ml methanol. Specific activity of the enzyme was determined by monitoring the hydrolysis of III by HPLC as in Example 5.

The maximum reaction rate for the enzyme was reached at 34° C. Little enzyme activity was found below 10° C and above 50° C.

EXAMPLE 7

Optimum salt concentration Test reactions were carried out similar to

Example 6 except that buffer concentrations ranging from 10 to 200 mM at 32° C were examined. All other conditions and analyses were the same.

As is apparent in Table 5, high salt

concentration markedly improved the specific activity of the enzyme. The effect was of a general nature and did not appear to be dependent on specific anions or cations.

EXAMPLE 8

Stability of phthalyl amidase

A. Effect of ionic strength

The stability of phthalyl amidase at pH values ranging from 6-9 was determined as described in Example 5 at 30° C in both 20 and 200 mM potassium phosphate buffer. In 20 mM buffer, all enzyme activity was lost within 2 hours at any pH of the incubation medium. In 200 mM buffer, the enzyme retained at least 80% of its activity for 100 hours irrespective of the pH of the incubating medium. Twenty mM buffer that was supplemented with 200 mM KCl or NaCl also protected against activity loss,

indicating that the enzyme stabilization was primarily dependent on the high ionic strength of the buffer. B. Temperature stability

The phthalyl amidase enzyme was also tested for stability at varying temperatures. The enzyme was

incubated at pH 8.2 in the temperature range of 4-50° C for 48 hours in 50 and 200 mM phosphate buffer. In 50 mM buffer, the enzyme retained 90% of its activity for 48 hours when maintained at temperatures below 25° C, while all enzyme activity was lost within 48 hours when the incubation temperature was above 40° C. In 200 mM buffer, 80% of the enzyme activity was retained in temperatures up to 35° C and 30% of the enzyme activity was retained after 48 hours incubation at 40° C.

EXAMPLE 9

Influence of effectors on enzyme activity

The effect of various agents on the enzymatic activity of phthalyl amidase was determined using standard conditions (see Example 5). All agents were tested at 1 mM final concentration unless otherwise indicated.

It can be seen from the data in Table 6 that iodoacetate, p-HMB, and copper ions significantly reduced phthalyl amidase activity. None of the tested compounds stimulated enzyme activity significantly above that of the control.

Table 7 shows the effects of four organic solvents at three concentrations on enzyme catalysis. All four solvents tested significantly decreased enzyme activity at a concentration of 10%. Glycerol caused the least amount of inhibition of the enzyme at the highest concentration tested.

DTT: dithiothreitol

p-HMB: para-hydroxy mercuric benzoate

DTNB: dithionitrobenzoate

NEM: N-ethylmaleimide

NAD: nicotinamide adenine nucleotide

NADP: nicotinamide adenine dinucleotide phosphate

NADPH: reduced form of NADP

ATP: adenosine 5 '-triphoεphate

PLP: pyrιdoxyl-5-phosphate

THF: tetrahydrofolate

FAD: flavin adenine dinucleotide

EXAMPLE 10

Physical and chemical properties of phthalyl amidase

The molecular weight of the phthalyl amidase was determined to be 49,900 by electrospray mass spectrometry. The enzyme is monomeric with an isoelectric point estimated by isoelectric focusing to be pH 5.5. Chemical hydrolysis and amino acid analysis of the protein by standard methods are shown in Table 8. Repeated attempts to sequence the N- terminus of the purified enzyme failed, indicating that the enzyme was blocked.

EXAMPLE 11

Substrate specificity of phthalyl amidase

A. Chemical structure requirements for enzyme activity

The activity of phthalyl amidase against 25 compounds was determined. The compounds were divided into beta-lactams (Table 9), phthalyl amides (Table 10), and aromatic ring substituted amides (Table 11). Each reaction mixture (1 ml total volume) contained 2.5 μmol of compound and 0.3 units of enzyme (based on the chromogenic

substrate) of the preparative scale purified enzyme, in 50 mM phosphate buffer, pH 8.0. The reactions were conducted at 30° C. Samples of the reaction mixture were taken at various times, and methanol (equal volume) was added to stop the reaction. The samples were examined by HPLC to determine the extent of substrate hydrolysis. The amount of compound hydrolyzed was calculated from a standard curve of the test compound. All substrates were stable in buffer at 30° C and pH 8.0 in the absence of enzyme for 24 hours.

As the results in Table 9 indicate, the enzyme recognizes mono- and bicyclic beta-lactam compounds containing a phthalyl group attached to the exocyclic nitrogen. However, the side chain apparently requires a 2- carboxylate group, for example, phthalate, since no hydrolysis is observed in the absence of this functional group.

A wide variety of phthalyl amides are substrates for the enzyme as shown in Table 10. Substrates include phthalylated amino acids, dipeptides, monocyclic and bicyclic beta-lactams, phenyl, benzyl, and aliphatic amines. The enzyme also exhibited esterase activity as demonstrated by its ability to hydrolyze phthalate mono methyl ester (IX). In this series, compound XIII was the most active compound found.

Using compound XIII as a standard, a variety of aromatic ring substituted compounds were examined for reactivity with the enzyme. Results are shown in Table 11. Aromatic ring substituents at the 6 position of the

phthalyl ring such as F and NH₂ were accepted by the enzyme. A hydroxyl group at the 3 position (XXI) of the ring and a nitrogen within the aromatic ring (XX) is also acceptable. Low levels of hydrolysis occur if a tetrazole is substituted for the 2-carboxylate group (XXII). Moving the carboxylate group to the 3 (XXIV) or 4 (XXIII) position of the aromatic ring completely eliminates hydrolytic activity. Compounds lacking the 2-carboxylate (XXV - XXVIII) are not suitable substrates for the enzyme.

These results are consistent with the enzyme being a novel catalyst that removes phthalyl protecting groups from a variety of amines under mild conditions.

B. Kinetic parameters for phthalyl amidase

The kinetic parameters of the enzyme were determined for several representative substrates. Compounds II, XVII, and XVIII were tested using 0.9 μg/ml of enzyme. Compounds III and XI were tested using 5.14 μg/ml of enzyme. Substrate concentrations were between 0 and 25 mM and reaction time was between 2 and 20 minutes, depending on the substrate used. All reactions were run at 32° C and at pH 8.2. The K_m, V_max, K_cat, and K_cat/K_m for these substrates are shown in Table 12. K_m is the Michaelis constant for enzyme kinetics, V_max is the maximal rate of reaction calculated by the Michaelis-Menten equation, and K_cat is the catalytic constant for an enzyme reaction.

a - carbacepham nucleus (7-amino-3-chloro-4-carboxy-1- carba-dethioceph-3-em ) (XXXIV) quantitatively monitored as the product of substrate III.

b - for the other substrates, phthalic acid was the product monitored during the reaction.

C. Chiral and additional substrate selectivity of phthalyl amidase.

Several additional substrates were tested in a total volume of 1 ml. Reaction mixtures contained 0.009 mg

(0.6 units) of enzyme, 2.5 μmol of substrate, and buffer A.

All reactions were run at 30° C for 2 minutes except for compounds XXX and XXXII, which were run for a longer time period since they were poor substrates for the enzyme.

Reactions were stopped by the addition of methanol, and phthalic acid formation was monitored by HPLC. Results are shown in Table 13. The results show that the enzyme has a marked preference for the D isomer of N-phthalyl-phenylglycine. The L isomer was an extremely poor substrate for the enzyme. Compound XXXI had a relative activity twice that of compound III as a substrate for the enzyme. However, by substituting a sulfonate group for the carboxyl group of the phthalyl moiety, enzyme reactivity is completely lost. Again, these results show the selectivity of this enzyme for N-phthalylated amines and indicate that the enzyme has a chiral preference on the amine side of the substrate.

EXAMPLE 12

Preparative scale synthesis of carbacephem nucleus

Phthalimido carbacephem (XXXIII) readily hydrolyzes to phthalamido carbacephem (III) in buffer at pH 8.0. Thus, either compound XXXIII or III can be used to prepare the carbacephem nucleus (XXXIV). Substrate (4 grams) was added to 20 ml of deionized water and the pH of the solution was adjusted to 8.0 with concentrated ammonium hydroxide. Phthalyl amidase, 80 units as determined using the chromogenic substrate (II), was added to start the reaction. Temperature was maintained at 30° C and the pH maintained at 8.0 by adding 2 N ammonium hydroxide. After 510 minutes under these conditions, HPLC analysis of the samples from the reaction indicated that compound III was 90.0% hydrolyzed and compound XXXIII was 98% hydrolyzed. The pH of the reaction was lowered to 5.0 thereby

precipitating the carbacephem nucleus. Isolated yields of the nucleus were between 65% and 77%.

EXAMPLE 13

Expression of met-phthalyl amidase in Escherichia coli

Several small scale temperature inductions of E. coli DH5α/pZPA400 were carried out to assess the amount of met-phthalyl amidase protein and enzymatic activity generated by E. coli DH5α/pZPA400. Enzymatic activity was observed by incubation of a soluble cell extract with the chromogenic substrate (II) under conditions as described in Example 3 or with substrate III as described in Example 5. Results are reported in Table 14.

SDS-PAGE gels of the cell extract showed a

Coomassie-stained protein band corresponding to

approximately 50,000 daltons that increased upon

temperature induction. Partial purification of the cell extract by anion exchange chromatography yielded fractions with increased phthalyl amidase specific activity.

Phthalyl amidase in these fractions catalyzed cleavage of the phthalyl group from compound III to form compound XXXIV and phthalic acid.

EXAMPLE 14

Expression of pro-phthalyl amidase open reading frame in

Streptomyces lividans

A 5 ml inoculum of Streptomyces lividans

TK23/pZPA600 (grown for 48 hours at 30° C, 280 rpm) was added to each of two 2 L shake flasks containing 500 ml Trypticase Soy Broth medium and cultured at 30° C, 280 rpm for 24 hours. Incubations beyond 24 hours were deleterious to production of phthalyl amidase. Cells were removed by centrifugation (4° C, 15 min, 12,000 x g) and phthalyl amidase activity in the cell-free broth was determined with compound III as substrate as in Example 13 (Table 14). The cell-free broth (800 ml, 0.10 mg/ml) was passed at 1 ml/min through a Mono Q column (10 x 10 mm (8 ml); Pharmacia). A linear gradient of 0 to 1.5 M KCl in buffer A was passed over the column and 2 ml fractions were collected. Most of the phthalyl amidase activity eluted in fractions 19 and 20 (about 0.75 M KCl).

A 1 ml aliquot of fraction 19 was concentrated 10-fold via ultrafiltration and analyzed by SDS-PAGE. A major protein band was observed at about 50,000 daltons, which corresponded to the molecular weight observed by electrospray mass spectrometry for purified mature phthalyl amidase obtained from Xanthobacter agilis. It also corresponded closely to the theoretical molecular weight predicted for a protein encoded by SEQ ID NO: 1.

Specific activity - units of enzyme/mg total protein

Volumetric activity - units of enzyme/liter of whole broth

Unit of enzyme - amount of enzyme that converts one nanomole of substrate in one minute

Phthalyl amidase is cell-bound for both Xanthobacter agilis and Escherichia coli; for Streptomyces lividans phthalyl amidase is secreted into the culture medium.

a - Method of release by sonication as in Example 4A. See also Example 4B where method of release by lysozyme treatment.

b - Method of assay as in Example 5. EXAMPLE 15

Use of recombinant phthalyl amidase to remove the phthalyl blocking group from phthalamido carbacephem Activity was assayed by the addition of phthalyl amidase (30 μl of Mono Q fraction 19 from Example 14, 1.83 μg total protein) to 1.82 μg of compound III in a 1 ml reaction mixture buffered by 200 mM potassium phosphate, pH 8.2. The reaction was carried out at 32° C for 20 minutes and stopped with the addition of 1 ml methanol. After removal of precipitate by centrifugation, an aliquot (30 μl) of the supernatant fraction was monitored by HPLC (254 nm absorbance) for both the carbacephem nucleus (XXXIV) and phthalic acid using a Zorbax C8 column (0.46 x 15 cm;

MacMod Analytical Inc.). The reaction products were eluted by a mobile phase constructed as continuous mixed gradients from (a) 1% acetonitrile/0.2% trifluoroacetic acid and (b) 80% acetonitrile/0.2% trifluoroacetic acid. The above substrate, loracarbef nucleus, and phthalic acid eluted at 11.0, 3.4, and 5.9 minutes, respectively. HPLC peaks were identified and quantitated using data generated by known amounts of authentic compounds. The specific activity of recombinant phthalyl amidase derived from fraction 19 for conversion of substrate was 9.5 μmol/min/mg protein.

EXAMPLE 16

Use of recombinant phthalyl amidase to remove the phthalyl blocking group from phthalimido-aspartame

In the synthesis of aspartame, the bivalent protection of the amino group of L-aspartic acid via a phthalimido moiety gives a superior substrate for a lyase catalyzed condensation with L-phenylalanine methyl ester. However, an efficient method to convert the phthalimido- protected compound back to the amine was previously

lacking. Following the condensation reaction, mild base was used to open the phthalimido moiety to a phthalamido moiety and recombinant phthalyl amidase was then used to catalyze hydrolysis of the latter to aspartame and phthalic acid (see Table 10).

Claims

We Claim:

1. A DNA compound that comprises an isolated nucleotide sequence encoding SEQ ID NO: 2.

2. The DNA compound of claim 1 wherein said isolated nucleotide sequence is SEQ ID NO: 1.

3. The DNA compound of claim 1 that further comprises a signal peptide-encoding nucleotide sequence located adjacent to and upstream from said isolated nucleotide sequence.

4. The DNA compound of claim 3 wherein said signal peptide-encoding nucleotide sequence is endogenous to Xanthobacter agilis.

5. The DNA compound of claim 3 wherein said signal peptide-encoding nucleotide sequence is SEQ ID NO: 8

6. The DNA compound of claim 1 that further comprises a transcriptional termination sequence adjacent to and downstream from said isolated nucleotide sequence.

7. The DNA compound of claim 6 wherein said transcriptional termination sequence is SEQ ID NO: 10.

8. A DNA compound that comprises an isolated nucleotide sequence encoding SEQ ID NO: 4.

9. The DNA compound of claim 8 wherein said isolated nucleotide sequence is SEQ ID NO: 3.

10. The DNA compound of claim 8 wherein said isolated nucleotide sequence is SEQ ID NO: 5.

11. The DNA compound of claim 8 wherein said isolated nucleotide sequence is SEQ ID NO: 6.

12. The protein, in purified form, having the amino acid sequence of SEQ ID NO: 2.

13. The protein, in purified form, having the amino acid sequence of SEQ ID NO: 4.

14. A recombinant DNA vector that comprises said isolated nucleotide sequence of claim 1.

15. The recombinant DNA vector of claim 14 that further comprises a promotor and translational activating sequence positioned to drive expression of said isolated nucleotide sequence.

16. The recombinant DNA vector of claim 15 wherein said promotor is endogenous to Xanthobacter agilis .

17. The recombinant DNA vector of claim 15 wherein said promotor is SEQ ID NO: 7.

18. The recombinant DNA vector of claim 15 wherein said isolated nucleotide sequence is SEQ ID NO: 1.

19. The recombinant DNA vector of claim 15 wherein said isolated nucleotide sequence is SEQ ID NO: 3.

20. The recombinant DNA vector of claim 15 wherein said isolated nucleotide sequence is SEQ ID NO: 5.

21. The recombinant DNA vector of claim 15 wherein said isolated nucleotide sequence is SEQ ID NO: 6.

22. The recombinant DNA vector of claim 15 that is plasmid pZPA600.

23. The recombinant DNA vector of claim 15 that is plasmid pZPA400.

24. A host cell transformed with said DNA vector of claim 15.

25. A host cell transformed with said DNA vector of claim 18.

26. A host cell transformed with said DNA vector of claim 19.

27. A host cell transformed with said DNA vector of claim 20.

28. A host cell transformed with said DNA vector of claim 21.

29. A host cell transformed with said DNA vector of claim 22.

30. A host cell transformed with said DNA vector of claim 23.

31. The host cell of claim 24 selected from the group consisting of Escherichia coli and Streptomyces lividans .

32. The host cell of claim 31 that is Streptomyces lividans/pZPA600 .

33. The host cell of claim 31 that is Escheri chia coli/pZPA400.

34. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 24 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

35. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 25 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

36. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 26 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

37. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 27 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

38. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 28 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

39. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 29 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

40. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 30 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

41. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 31 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

42. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 32 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

43. A method for producing recombinant phthalyl amidase, said method comprising culturing a host cell of claim 33 under conditions suitable for gene expression, and recovering the expressed phthalyl amidase from the culture.

44. The DNA compound of claim 1 wherein said isolated nucleotide sequence is SEQ ID NO: 11.

AMENDED CLAIMS

[received by the International Bureau on 13 December 1995 (13.12.95);

original claims 24-33 amended; new claims 45-49 added;

remaining claims unchanged (5 pages)]

1. A DNA compound that comprises an isolated

nucleotide sequence encoding SEQ ID NO:2.

2. The DNA compound of claim 1 wherein said

isolated nucleotide sequence is SEQ ID NO: 1.

3. The DNA compound of claim 1 that further

comprises a signal peptide-encoding nucleotide sequence located adjacent to and upstream from said isolated

nucleotide sequence.

4. The DNA compound of claim 3 wherein said signal peptide-encoding nucleotide sequence is endogenous to

Xanthobacter agilis.

5. The DNA compound of claim 3 wherein said signal peptide-encoding nucleotide sequence is SEQ ID NO: 8.

6. The DNA compound of claim 1 that further

comprises a transcriptional termination sequence adjacent to and downstream from said isolated nucleotide sequence.

7. The DNA compound of claim 6 wherein said

transcriptional termination sequence is SEQ ID NO: 10.

8. A DNA compound that comprises an isolated

nucleotide sequence encoding SEQ ID NO: 4.

9. The DNA compound of claim 8 wherein said

isolated nucleotide sequence is SEQ ID NO: 3.

10. The DNA compound of claim 8 wherein said

isolated nucleotide sequence is SEQ ID NO: 5.

13. The protein, in purified form, having the amino acid sequence of SEQ ID NO:4.

16. The recombinant DNA vector of claim 15 wherein said promotor is endogenous to Xanthobacter agilis.

20. The recombinant DNA vector of claim 15 wherein said isolated nucleotide sequence is SEQ ID NO:5.

22. The recombinant DNA vector of claim 15 that is plasmid pZPA600.

23. The recombinant DNA vector of claim 15 that is plasmid pZPA400.

24. A bacterial host cell transformed with said DNA vector of claim 15.

25. A bacterial host cell transformed with said DNA vector of claim 18.

26. A bacterial host cell transformed with said DNA vector of claim 19.

27. A bacterial host cell transformed with said DNA vector of claim 20.

28. A bacterial host cell transformed with said DNA vector of claim 21.

29. A bacterial host cell transformed with said DNA vector of claim 22.

30. A bacterial host cell transformed with said DNA vector of claim 23.

31. The bacterial host cell of claim 24 selected from the group consisting of Escherichia coli and

Streptomyces lividans.

32. The bacterial host cell of claim 31 that is

Streptomyces lividans/pZPA600.

33. The bacterial host cell of claim 31 that is Escherichia coli/pZPA400.

44. The DNA compound of claim 1 wherein said

isolated nucleotide sequence is SEQ ID NO: 11.

45. A DNA compound that comprises an isolated

mucleotide sequence which is SEQ ID No: 7.

46. A DNA compound that comprises an isolated

mucleotide sequence encoding SEQ ID No: 9.

47. The DNA compound of claim 46 wherein said

isolated mucleotide sequence is SEQ ID No: 8.

48. An amino acid sequence according to SEQ ID No: 9.

49. An amino acid sequence according to SEQ ID No: 12.

STATEMENT UNDER ARTICLE 19

The International Search Report for the captioned application was mailed 13 October 1995. In accordance with Article 19 ofthe Patent CooperationTreaty, applicant submits herewith five replacement sheets (pages 77-81) for pages 77-81 of the International Application as originally filed.

Claims 24-33 have been amended to identify the host cell as a bacterial host cell. Claims 45-49 have been added and are directed to alternative embodiments of the present invention.

In accordancewith Article 19(2), the amendments do not go beyond the disclosure of the international application as filed. Applicant respectfully requests entry of the substitute sheets for pages 77-81 of the International Application as originally filed.